NMDA AMPA Receptors
Excessive excitation by neurotransmitters can cause the degeneration and death of neurons. It is believed that this degeneration is in part mediated by the excitotoxic actions of the excitotoxic amino acids (EAA) glutamate and aspartate at the N-methyl-D-aspartate (NMDA) receptor, the alpha-amino-3-hydroxy-5-methyl 4-isoxazole proprionic acid (AMPA) receptor, and the kainate receptor. AMPA/Kainate receptors may be referred to jointly as non-NMDA receptors.
This excitotoxic action is considered responsible for the loss of neurons in cerebrovascular disorders such as cerebral ischemia or cerebral infarction resulting from a range of conditions, such as thromboembol or hemorrhagic stroke, cerebral vascospasm, hypoglycemia, cardiac drowning, pulmonary surgery, and cerebral trauma, as well as Alzheimer's disease, Parkinson's disease, and Huntington's disease.
Among excitatory amino acid receptor antagonists recognized for usefulness in the treatment of neurological disorders are those that block AMPA receptors. (Bigge C. F. and Malone T. C., Curr. Opin. Ther. Pat., 1993:951; Rogawski M. A. TiPs, 1993; 14: 325).
AMPA receptor antagonists have prevented neuronal injury in several models of global cerebral ischemia. (Li H. and Buchan A. M., J. Cerer. Blood Flow Metab., 1993; 13: 933; Nellga B. and Wielock T. J. Cerer. Blood Flow Metab., 1992; 12:2) and focal cerebral ischemia (Bullock R., Graham D. I. Swanson S., McCullock., J. Cerer. Blood Flow Metab. 1994; 14: 466; Xue D. et al J. Cerer. Blood Flow Metab., 1994; 14: 251).
AMPA receptor antagonists have also demonstrated promise in chronic neurodegenerative disorders such as Parkinson's disease. (Klockgether T. et al., Ann. Neurol., 1993; 34 (4): 585-593).
Excitatory amino acid receptor antagonists that block NMDA receptors are also recognized for usefulness in the treatment of disorders. NMDA receptors are intimately involved in the phenomenon of excitotoxicity, which may be a critical determinant of outcome of several neurological disorders. Disorders known to be responsive to blockade of the NMDA receptor include cerebral ischemia (stroke or cerebral trauma, for example), muscular spasm, convulsive disorders, neuropathic pain, and anxiety, and may be a significant causal factor in chronic neurodegenerative disorders such as Parkinson's disease (Klockgether T., Turski L., Ann. Neurol. 1993; 34: 585-593), human immunodeficiency virus (HIV) related neuronal injury, amyotrophic lateral sclerosis (ALS); Alzheimer's disease (Francis P. T, et al. J. Neurochem. 1993; 60(5): 1589-1604) and Huntington's Disease (Lipton S., TINs, 1993; 16(12): 527-532; Lipton S., Rosenberg P. A. New Eng. J. Med 1994; 330 (9): 613-622); Bigge C. F. Biochem. Pharmacol. 1993; 45: 1547-156).
NMDA receptor antagonists may also be used to prevent tolerance to opiate analgesisa or to help control withdrawal symptoms from addictive drugs.
A Novel Antagonist to NMDA/AMPA
This invention relates to the discovery of the functions of cyclic Propyl Glycine (herein referred to as “cyclic PG” or “cPG”) as a novel antagonist that either blocks the AMPA and/or the NMDA receptors.
CPG has been found to be an endogenous compound exhibiting anxiolytic activities in animal studies by Gudasheva T. A et al (Biull Eksp Biol Med 1999 October 128:10 411-3) and Seredenin S. B. et al (Bull Exp Biol Med 2002 April 133:360-2).
We have surprisingly discovered that cPG is the end product of the metabolites of glycine-proline-glutamate (GPE), which in turn is one of the components of the cleavage of insulin-like growth factor I (IGF-I).
IGF-I is a 70 amino acid-long polypeptide with several metabolic actions known to be expressed in the rat brain during development and after acute injury. (D'Ercole, A J et al Molecular Neurobiology 1996; 13: 227-255).
Some of the biological effects of IGF-I are probably facilitated by des (1-3) IGF-I, an IGF-I derivative lacking the N-terminal tripeptide glycine-proline-glutamate (GPE). It was reported that des (1-3) IGF-I is less effective than recombinant human IGF-I (nhIGF-I) as a neuronal rescue agent, which suggests that the central effect of IGF-I might be partially mediated by the tripeptide GPE. (Guan J, et al Endocrinology 1996; 137: 893-898).
However, it was Sara et al, who in 1989 showed that GPE is a neuroactive peptide which facilitates the release of both acetylcholine and dopamine from cortical slices in vitro. (Biochem. Biophys. Res. Comn 1989; 165: 766-771).
Sarah's group has Swedish, European, and Japanese patents on GPE as a neuromodulatory peptide (EP0366638, SE8803847, JP2250895).
The US patent by Bourguignon et al, U.S. Pat. No. 5,804,550 or W094/26301 suggests that GPE is an NMDA antagonist.
The US patent by Gluckman et al, U.S. Pat. No. 6,187,906 claims that GPE can be used to protect dopaminergic neurons of a mammal against death from Parkinson's disease. This group reported that the mechanism by which GPE leads to prevention of cell death was not known, but was not by modulating neuronal activity.
In their patent claims, the Gluckman group proposed that the “concentration of GPE and/or analogues thereof in the CNS and in the brain of the patient in particular should be increased in order to treat the CNS damage.” They also proposed a suitable dosage range to be between about 0.1 to 1,000 μg of GPE per 100 g of body weight where the composition is administered centrally.
However, the Gluckman patent (filed on Jul. 15, 1999 and issued on Feb. 13, 2001) did not cite a publication by Curatolo L. et al (Annals of New York Academy of Sciences 1995; 765: 145-150 Neuroprotective effect of GPE Pretreatment on rat Hippocampal Organotypic cultures exposed to NMDA), in which the effect of GPE did not seem to be clearly concentration-dependent. The highest degree of neuroprotection was obtained with 10 μM GPE while lower (1 μM) or higher (50-100 μM) concentrations reduced the neuronal damage to a lesser extent. The lack of a concentration-dependent effect does not indicate a single receptor-mediated effect. In fact, the bell-shaped curve describing the pharmacological effect of increasing concentrations of GPE might be the result of multiple mechanisms of action.
Without the clear understanding of the mechanism of action of GPE, it was very difficult to determine the optimal dosage or human treatment. The empirical bell-shaped curve-model for rats may be only applied to rats and might not be entirely appropriate for human applications.
Private communication with the Gluckman group showed unpublished results of recent studies in which high concentrations of GPE administered intraperitoneally and intravenously caused severe brain damage to rats. It was questionable whether GPE may be a suitable neuroprotective agent based on unreproducible results of the bell-shaped curve concentrations of GPE.
The uncertainty of the concentration dependency was not only observed in GPE, but also with IGF-I. Johnston B. et al (J. Clini. Invest. Volume 97, Number 2, January 1996, pp 300-308 and subsequent private communications) reported that 1 μg IGF-I is more neuroprotective than 100 ng in fetal sheep studies, but when the dose increased from 1 μg to 10 μg all the neuroprotective effects are lost. It was found that increasing the dose to 100 μg or more usually killed the sheep fetus. It was not known why the neuroprotective dose in the fetal sheep (weighs about 3.5 kg) was about 500 times less than the effective dose in the rat (weighs about 350 g) if the difference in weight between the fetus and rat is taken into account (a fetal sheep weighs about 10 times the rat).
The present invention provides an explanation to the unpredictable bell-shaped curve effect of the concentrations of IGF-I and GPE. The present invention provides a novel mechanism of action of GPE, in which GPE is not a final product of the cleavage of IGF-I, but GPE is metabolized into cyclic Propyl Glycine and glutamic acid, as illustrated in FIG. 1.
Over the past twenty years there have been a large number of publications reporting the neurological effects of the insulin like growth factors. (Pimentel E. (1994) Handbook of Growth Factors, Volume 1-3, CRC Press (Ann Arbor). Now with the finding that IGF-1 and GPE are the pro-drug of cPG, we now can link the biological activities of these pro-drugs to cPG, thus not only enabling us to study the mechanisms of action of IGF-1 and GPE, but also to attribute the neuroprotective activities of these two compounds to cPG and their neurotoxicity to the corresponding glutamic acids.
It is noted that the stereoisomers of GPE are important factors and it is found that only the cis form of GPE can be metabolized into cyclic Propyl Glycine and glutamic acid. Since glutamic acid or glutamate is a well-known neurotoxic agent, it is predicted that cyclic Propyl Glycine (cPG) must possess very potent neuroprotective effect to overcome the intrinsic neurotoxicity of glutamate.
As shown in the FIGS. 1 and 2, cyclic PG can form chelating complexation with metal ions such as calcium ions, magnesium ions, and it also can bind to large molecules such as IGF-Binding Proteins, such that cyclic PG can serve as a neurotransmittance agent or a neurotransportor as well as an energy storage in the central nervous system. These attributes can make cyclic PG an anti-necrotic and anti-apoptotic agent in the central nervous system.
Our experimental results reported herein, serving as limited examples, showed that cPG not only acts as a potent neuroprotective agent but also serves as a neurogenesis agent, which can be considered a novel drug candidate for treatment of neurological disorders.